Pichia pastoris and E. coli expression systems are suitable to express products with a molecular weight of about 5 to 200 KD. Both of these two systems can not express short peptides with a molecular weight of about 1 to 5 KD, or even if some peptides can be expressed, the expression levels are relatively low in general.
In addition, there is another problem for the expression of short peptides with low molecular weights, i.e., it is difficult to detect the products. The normal molecular weights of standards in the conventional SDS-PAGE are between the range of 14-65 KD. Short peptides are out of the detectable range (the highest concentration of gels used in SDS-PAGE is about 20%, where the detectable linear range of the molecular weight is 10 to 40 KD); short peptides are, therefore, prone to run out of the gel, leading to imprecise results.
Short peptides can be used to treat a lot of common diseases. For example, diabetic nephropathy (DN) is one of the chronic common complications of diabetes, and has become a major cause for end-stage renal disease (ESRD). About 35% type I diabetic patients and 15% type II diabetic patients will finally develop diabetic nephropathy. The proinsulin C-peptide is a peptide secreted by the pancreatic beta cells, which consists of 31 amino acid residues. It has been demonstrated that the proinsulin C-peptide can ameliorate long-term diabetic complications (including diabetic nephropathy). Furthermore, it has also been shown that the C-peptide, employed alone or together with insulin, can reduce the mesentery expansion resulted from extracellular matrix accumulation around renal glomeruli. This function of the C-peptide may explain the major mechanism for treating diabetic microangiopathy, including diabetic nephropathy. Accordingly, under the circumstance that insulin still moderately regulates carbohydrate metabolism, a combination treatment with the C-peptide can significantly ameliorate the onset and development of diabetic nephropathy. Since there are millions of diabetic patients, short peptide drugs such as the C-peptide are in great demand.
At present, short peptide drugs used in the international market are mostly produced by chemical synthesis with high cost. Therefore, there is an urgent need for genetic engineering methods to produce short peptides on a large scale and with simple process and low cost.
Constructing multiple copies is an efficient means to increase the expression level of short peptides. Multicopy genes can be constructed to express fusion proteins with high molecular weight, so as to achieve high efficient expression and facilitate detection.
For multicopy fusion proteins with high expression efficiency and the genes encoding the same, the following problems must be addressed: (a) selection of the target gene (or target protein); (b) length (i.e., the number of copies); (c) ligation linker, said ligation includes the ligation of fusion protein with the expression plasmid, the ligation between target protein monomers, and the selection of signal peptides, etc.; and (d). enzymatic digestion after expression. Any inappropriate selection would affect the expression level, or even prevent the fusion protein from expressing.
One common strategy is to fuse the multicopy short peptide with another peptide having higher molecular weight (about 10-20 KD) to ensure the stable presence of the fusion protein in host cells. The disadvantage of this method, however, is that the desired short peptide only accounts for a small proportion in the fusion protein, thus lowering the efficiency. Furthermore, fusion proteins formed with GST or other proteins add to the processing complexity, which requires the step of enzymatic digestion and purification.
Another ideal strategy is to insert some short upstream and downstream sequences at the upstream or downstream of the multicopy short peptide, which significantly increase the proportion of the short peptide in the fusion protein. Yet at present, most of the fusion proteins constructed by this method are unstable and can not yield expression products in a large amount.
Accordingly, there is an urgent need in the art to develop a method for expressing multicopy short peptides high effectively and stably.